476 Effect of Holding Period on Viability, Acrosome Status and Intracellular Zinc of Boar Sperm During Liquid Storage

Abstract Sperm capacitation results in acrosomal remodeling, increased membrane fluidity, plasma membrane fusability and hyperactivated sperm. Distinct sperm zinc signatures reflect intracellular zinc content, capacitation status, and viability. Therefore, flow cytometric assessment could be a useful tool to evaluate sperm quality and longevity during liquid storage of boar semen. Cooled semen (17°C) from commercial boars (n = 12) were assessed at Day-1, -4 and -7 after collection for motility (IVOS II, Hamilton Thorne, Beverly, MA), intracellular zinc and viability. Samples were stained with 8 µg/mL Hoecsht-33342 (Hoe), 1 µM FluoZinTM-3-AM (Zinc), 10 µM propidium iodide (PI) and 5.6 µg/mL PNA-Alexa647TM (Molecular Probes, Eugene, OR), incubated for 30 min, washed with PBS, and aditionally incubated for 30 min. Acquisition and analysis of 20000 events were performed using Bio-Rad ZE5 Cell Analyzer (Hercules, CA) and FLowJoTM (Ashland, OR). Hoe+ events were gated into Zinc/PI plot to determine viable sperm with high Zinc (Zinc+/PI-). Likewise, Hoe+ events were gated into PI/PNA plot and then into Zinc histogram to detect viable-intact-acrosome with high Zinc (Zn+/PI-/PNA-). Data were analyzed by mixed model for repeated measures, Tukey-adjusted pairwise comparisons and Pearson’s correlation. Viable sperm at Day-1 (86.0±3.1; emmean±SM) and Day-4 (84.7±2.8) showed similar percentage of Zinc+ sperm, and both significantly higher compared to Day-7 (52.2±2.8). Also, viable-intact-acrosome sperm displayed similar percentage of Zinc+ sperm at Day-1 (93.8±3.4) and Day-4 (91.6±3.4) and both higher than sperm at Day-7 (64.5±3.1; P < 0.0001). Percentages of Zinc+ sperm were higher (P < 0.001) when viable sperm had intact acrosome within Day. Percentages of Zinc+ sperm strongly correlated to viable-intact-acrosome sperm (r=0.76, P < 0.0001) and total motility (r=0.65, P < 0.0001). We conclude that sperm having intact plasmatic membrane and acrosome display higher intracellular zinc content which is related to capacitated status, remaining non-capacitated until at least to Day-4 after collection in the tested extender.